1. Technical Field
The present invention is related to a plasmid having mini Mu transposons and a method of rapid DNA (deoxyribonucleic acid) sequencing. More particularly, the present invention is directed to the use of random insertion of transposons as a part of rapid and convenient process related to DNA sequencing.
2. Prior Art
Since the development of DNA sequencing techniques, several refinements and improvements have occurred in sequencing methodology and there are a number of approaches available for different sequencing requirements.
The most critical step in sequencing method is the set of four nucleotide specific sequencing reactions. Each one of the reactions produces a group of radioactively-labelled DNA fragments, one end of which is fixed (starting point) and the other ends are distributed among the locations of one of the four nucleotides along the DNA. The products of the set of four reactions are then separated side by side by a gel electrophoretic method, which separates each DNA fragment according to its length, and visualized by autoradiography. The autoradiogram reveals the positions of each of the four nucleotides as the distance from the fixed (starting) point, thus the nucleotide sequence adjacent to the fixed point can be determined.
Two different methods are now available for the sequencing reaction: one is the nucleotide specific chemical modification and cleavage reactions of Maxam and Gilbert, PNAS, 74:560, 1977, and the other is the primer extension reactions in the presence of nucleotide specific chain terminators as described by Sanger et al., PNAS 74:5463, 1977. Although it has been used widely and is still an important method, the chemical method of Maxam and Gilbert, supra, is more suited for sequencing of relatively short and wel-defined DNA pieces because of its relatively labor-intense nature.
At present, the chain terminator method of Sanger et al., supra, has gained wide acceptance as the method of choice for determining the base sequence of a long stretch of unknown DNA.
Since the length of DNA sequence, that can be determined conveniently by one round of either of the two sequencing methods available at the present time, is limited to about 300 base pairs, one has to set up many rounds of sequencing reactions to determine the sequence of a large piece of DNA. Each round uses a different starting point along the DNA, preferably several hundred bases away from its neighbors. For the base specific chain terminator method of Sanger et al., supra, the starting point is the end of the primer sequence used, or rather the end of the unknown sequence that is joined artificially to a universal primer sequence on a proper cloning vector.
Thus, for the determination of a long stretch of unknown sequence, the first step necessary is to generate a library of clones of the DNA to be sequenced. Each member of the library must have a unique starting point for sequencing that is within the range of sequence determination from the neighboring starting point in another member of the library, so that the sequence segments overlap and can be assembled into one continuous piece.
An often used method presently known for determining the sequence of a long stretch of unknown DNA may be outlined as follows.
The DNA segment of interest is randomly fragmented into small pieces which are cloned into phage M13 cloning vectors; the viral DNA of each clone is then used as a template in combination with a synthetic universal sequencing primer oligonucleotide in the chain terminator sequencing reaction; the reaction products made in the presence of .sup.35 S-labelled substrate are separated by gradient gel electrophoresis, visualized by autoradiography and the sequence is analyzed and assembled with the help of computer means.
While the scheme just outlined is widely used, it has certain shortcomings. One of the major problems derives from the fact that the DNA segment to be sequence is first fragmented into small pieces (a few hundred base pairs). The preparation of random clones of the small pieces, although not an overwhelming task, is still an elaborate, complex and time-consuming step. Furthermore, there is no certainty that the set of clones used for sequencing would cover the entire length of the DNA. For instance, if at the step of sequence assembly a missing stretch is discovered, it is a tedious and complicated task to search the entire clone library for the missing clone(s) to fill in the missing link. This is because the information on the position of each subfragment within the original long DNA piece has been lost by the random subcloning process.
The present invention discloses a rapid and convenient method which inter alia solves the problems mentioned above by using random insertion of transposons, particularly a mini Mu transpon, into a large cloned piece of DNA, instead of the random cloning of small fragments.
Random insertions of the transposon can be done by very simple steps using in vivo reactions in Escherichia coli cultured cells. This brings a substantial time saving. Since the entire DNA segment of interest remains intact without fragmentation in small pieces as is required in the hitherto known conventional process it is easy to assess which part of the segment has been sequenced during the process by analyzing the mini Mu insertion site. This is a great advantage when the sequence of a large segment of DNA has to be determined.